Structure of recombinant ricin A chain at 2.3 Å

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple -stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the -stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H2O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.is the catalytic subunit of ricin, a Centers for Disease Control and Prevention category B bioterrorism agent derived from Ricinus communis seeds. RTA catalyzes the depurination of an invariant adenosine residue, A 4234, within the GA 4234 GA tetraloop motif of the highly conserved sarcin-ricin loop of eukaryotic 28S rRNA (1). Clinical trials have exploited the toxicity of RTA in RTA-antibody constructs to kill leukemia and lymphoma cells (e.g., RTA conjugated to anti-CD22) (2-5). Side effects limit the utility of RTA immunotoxins (6, 7). Targeted inhibitors against ribosome-inactivating proteins (RIPs) could improve immunotoxin cancer therapies by rescuing normal cells following toxin treatment.Saporin-L1 (SAP), a homologue of RTA from Saponaria officinalis (soapwort) leaves, exhibits N-glycohydrolase activities on 80S ribosomes, poly(A) RNA, and other cellular . SAP releases multiple adenines from ribosomes, whereas RTA shows exquisite specificity.Truncated oligonucleotide constructs of the ribosomal sarcinricin loop are RTA and SAP substrates (13-16). In addition to hairpin stem-loop structures, RTA hydrolyzes adenine from cyclic GAGA loops that possess a 5Ј-to 3Ј-covalently closed synthetic linker (17, 18) (Fig. 1).Transition state analysis of RTA-mediated depurinations established that hydrolysis of adenine involves a ribocation intermediate, followed by attack of an activated water. Adenine activation is a major driving force for RTA catalysis (19,20). Efficient catalysis by RTA requires the invariant Glu-177 and Arg-180 residues (21-25). RTA and other RIPs have evolved to become near-perfect catalysts for mammalian ribosomes (21,(26)(27)(28). Here, we use transition state analogues to establish the catalytic site features contributing to this remarkable catalytic activity.RTA transition state structures have guided the design and synthesis of potent RIP inhibitors (17, 29). Inhibitors for RTA and SAP include 1Ј-aza-sugars with a nonhydrolyzable 9-deazaadenine to mimic the...

Section: Discussionmentioning

confidence: 99%

Transition state analogues in structures of ricin and saporin ribosome-inactivating proteins

Sturm

Almo

et al. 2009

“…Localization of the (x)D(y) Motifs in RTA, PE38-lys, and IL-2. The LDV motif in RTA (residues 74-76) is at the C terminus of a ␤-strand of the first domain near the Tyr-80 residue, which is involved in building the active site (16) (Fig. 1A).…”

Section: Peptide Origin Designation Descriptionmentioning

confidence: 99%

Evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome

Baluna

Rizo

Gordon

et al. 1999

180

124

The dose-limiting toxicity of interleukin-2 (IL-2) and immunotoxin (IT) therapy in humans is vascular leak syndrome (VLS). VLS has a complex etiology involving damage to vascular endothelial cells (ECs), extravasation of f luids and proteins, interstitial edema, and organ failure. IL-2 and ITs prepared with the catalytic A chain of the plant toxin, ricin (RTA), and other toxins, damage human ECs in vitro and in vivo. Damage to ECs may initiate VLS; if this damage could be avoided without losing the efficacy of ITs or IL-2, larger doses could be administered. In this paper, we provide evidence that a three amino acid sequence motif, (x)D(y), in toxins and IL-2 damages ECs. Thus, when peptides from RTA or IL-2 containing this sequence motif are coupled to mouse IgG, they bind to and damage ECs both in vitro and, in the case of RTA, in vivo. In contrast, the same peptides with a deleted or mutated sequence do not. Furthermore, the peptide from RTA attached to mouse IgG can block the binding of intact RTA to ECs in vitro and vice versa. In addition, RTA, a fragment of Pseudomonas exotoxin A (PE38-lys), and fibronectin also block the binding of the mouse IgG-RTA peptide to ECs, suggesting that an (x)D(y) motif is exposed on all three molecules. Our results suggest that deletions or mutations in this sequence or the use of nondamaging blocking peptides may increase the therapeutic index of both IL-2, as well as ITs prepared with a variety of plant or bacterial toxins.

“…Helix E spans the hydrophobic core of the protein and positions two of the invariant residues, Glu-177 and Arg-180, in the active site at the bottom of the cleft. Helices D and E cross each other at an angle of =46°and interact through local hydrophobic forces (5)(6)(7)9). This interaction is not sequence specific or dependent on particular amino acid side chains per se because all 12 of the amino acids in helix D could be removed in one or another of the active mutants (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…RA has a prominent cleft (refs. [4][5][6][7][8][9] and Fig. 1) that contains residues that are conserved among a number ofplant and bacterial toxins (4,5) that share the RA mechanism of action (2,3).…”

mentioning

confidence: 99%

Analysis of the contribution of an amphiphilic alpha-helix to the structure and to the function of ricin A chain.

Morris

Wool²

1994

The A chain of ricin is a cytotoxic RNA Nglycosidase that inactivates eukaryotic ribosomes. The contribution of the amphiphic helix D, which Is distant from the active site, to the catalysis of the depurination of the adenosine at position 4324 in 28S rRNA has been examined by systematic deletion of amino acids. Two sets of consecutive two-or threeamino acid deletions of the 12 residues in helix D, a total of 20 mutants, were constructed. AU 12 of the amino acids could be deleted in one mutant or another without loss of activity; however, mutations that disrupted the amphiphic of the helix led to inactivation of the enzyme. Thus, the minimum contribution of helix D to the structure of the ricin A chain is to provide hydrophobic and hydrophilic surfaces to shield helix E, which has the active-site residues; moreover, no amino acid side chain in helix D makes a specific contribution to the recognition of the RNA substrate or to catalysis; and, finaliy, phasing of the amino acid deletions can be important to the phenotype of mutants.